Background: X-ray computed tomography (CT) is one of the most useful diagnostic tools for clinicians due to its widespread availability, fast scan times, and low cost. However, molecular imaging with CT is not possible with existing contrast agents, so clinicians must instead rely on other modalities which are more expensive and less readily available. Dual-energy CT (DE CT), a relatively new technique in which two x-ray energies are used for a single scan, can provide valuable information about tissue material composition. This information can potentially be used for molecular imaging if it is coupled with appropriately-designed contrast agents. The goal of this proposal is to develop for the first time an activatable DE CT contrast agent that can be used for the molecular imaging of protease activity. This goal will be met in the following specific aims: Aim 1: Develop and characterize in vitro a protease-cleavable nanoparticle CT contrast agent. Aim 2: Test in vivo performance of cleavable nanoprobes in healthy mice and in a mouse primary sarcoma model using dual energy CT. Methods: In Aim 1, a composite nanoprobe will be developed consisting of iodine-containing liposomes (~120 nm) linked to multiple small (~5 nm) gold nanoparticles by a protease-cleavable peptide. Stability, cleavage, and toxicity of the probe will be studied in vitro In Aim 2, this nanoprobe will undergo biodistribution/kinetics studies in healthy mice and then will be used for in vivo imaging of tumor protease activity in a mouse model of primary soft tissue sarcoma. When this probe is injected in vivo, tumor protease activity will cause cleavage of the peptide linking the gold nanoparticles to the liposome. After cleavage, the small gold nanoparticles will separate from the larger liposomes. Thus, in areas of low proteolytic activity, the DE CT gold and iodine signals should remain overlaid, whereas in areas of high proteolytic activity, the iodine signal should remain high while the gold signal will decrease. The measured ratio of gold to iodine concentration within tissues will be used to evaluate local protease activity. CT measurements of protease activity will be validated by protease-activatable optical probes and by ex vivo tissue analysis. Long-Term Objectives: This study will demonstrate the first use of a CT probe for molecular imaging of protease activity in cancer. This activatable probe has the potential to be used for CT imaging of multiple pathologies which exhibit high protease activity, including cancer, atherosclerosis, and other inflammatory conditions. Development of such a probe will be a significant step forward for non-invasive molecular imaging and has the potential to improve diagnosis and risk stratification for a variety of disease processes.